ABSTRACT There is a significant burden of malaria in young African infants - the majority of whom are breastfed from birth up to 24 months. These `break through' malaria infections probably indicate incomplete protection by breastfeeding. There is a significant gap in our knowledge about antimalarial immunity mediated by breastfeeding and the factors involved. Our recent preliminary data demonstrated that high concentrations of specific human milk oligosaccharides (HMOs) in Ugandan mothers' milk are differentially associated with malaria risk in their infants. HMOs represent the third most abundant component in breast milk which, upon ingestion by infants, reach the systemic circulation where they directly engage innate immune cells through specific surface receptors. Studies involving ex vivo stimulation of human cells and cell lines demonstrated that HMOs modulate the functions of macrophages and monocytes in several ways. This includes inducing anti-inflammatory or proinflammatory responses, inducing or inhibiting monocyte or macrophage activation, inhibition of proliferation or migration, and enhancing phagocytosis. A human clinical trial involving infants fed 2'- fucosyllactose (2'FL) in formula confirmed the anti-inflammatory effect of 2'FL. HMO-mediated modulation of monocyte function is highly relevant to pediatric malaria which involves monocyte dysregulation and systemic inflammation. Our long-term goal is to elucidate the role of HMOs in early innate immunity against malaria. The objective of this project is to determine the relationship between HMOs in breast milk and infant blood, monocyte function, and malaria outcomes in breast-fed infants. The central hypothesis is that direct HMO modulation of monocytes tips the balance towards disease or protection during pediatric malaria and that differences in levels of specific HMOs are linked to different malaria outcomes. This hypothesis will be tested in two Specific Aims. Aim 1: To recruit a birth cohort and prospectively collect health metadata including malaria outcomes and concentrations of soluble monocyte activation markers, cytokines and chemokines in infants. A birth cohort of 388 mother-infant pairs will be recruited and infants followed over 18 months with blood sampling every month and during infant illness for malaria diagnosis, multiplex bead-based cytokine and chemokine assays and ELISA for soluble monocyte activation markers. Aim 2: To measure concentrations of HMOs in mothers' milk and infant plasma to assess their association with monocyte mediators and malaria outcomes in infants. We will measure concentrations of HMOs in mothers' milk and infant plasma monthly and during pediatric malaria attacks to identify HMOs that are associated with specific monocyte inflammatory cytokines and chemokines and activation markers which are, in turn, linked to distinct malaria outcomes. To account for correlation between multiple measurements in the same infant, the relationships between malaria outcomes, specific HMOs, and markers of monocyte function will be assessed by Generalized Estimating Equation method while controlling for covariates such as maternal age and parity, infant sex, season of sampling and other malaria risk factors. The team brings experience in malaria and HMO research plus access to a study population in Uganda. Together, these proposed exploratory studies will provide preliminary insights on the crucial role that HMOs play in malaria innate immunity. This project will set the stage for more powered mechanistic studies of HMO-monocyte interactions which will inform the design of novel antimalarial interventions. The results are generalizable to other regions with a high burden of malaria in young infants. The potential to move the field forward and make a sustainable impact is high.